The long-term goal of this study is to understand the biological function of protein modification by the ubiquitin like protein ISG15. In recent years our knowledge of protein ubiquitination (ubiquitylation) has expanded rapidly and as a consequence protein ubiquitylation has been found to play important roles in various aspects of cellular function, including the cell cycle, membrane receptor signal transduction, endocytosis, protein quality control, transcription, and DNA repair. In contrast, little is known about the role of protein modification by ISG15. ISG15 is encoded by an interferon stimulated gene. Its expression is highly upregulated by Type I interferon and by bacterial and viral infections. Since ISG15 is not found in simple eukaryotic organisms, such as yeast and nematodes, it is unlikely to be a house keeping gene. Instead, it should be involved in specialized functions in complex organisms, such as human and mouse. In the past four year funding period, we have identified a group of ISG15 targets, characterized the effect of ISG15 modification on some of these targets, identified the ISG15 E2 and a few of the E3 enzymes, and established ISG15 E1 knockout mice. This proposal for the next two-year funding period will test the hypothesis that protein ISG15 modification plays an important role in modulating cellular function during immune responses. The studies proposed in Specific Aim #1 will identify ISG15 modification sites via high throughput screening of ISGylated proteins. The studies proposed in Specific Aim #3 will characterize ISG15 activating enzyme UBE1L knockout mice to examine molecular mechanisms of the function of protein ISGylation. These proposed studies will address important questions about protein ISGylation and may provide valuable insights into the prevention and therapeutic treatment of human diseases, such as pathogen infections and immune defects.